1. Field of the Invention
The present invention relates to a glass capillary for DNA analysis and a manufacturing method thereof, and to a DNA analyzer that uses the glass capillary.
2. Prior Art
One of the methods of analyzing DNA is electrophoresis. Electrophoresis has the advantages that, since laser-excited fluorescence is detected in real time, both the sensitivity and the throughput are high.
In a conventional DNA analyzer that uses electrophoresis, a plurality of glass capillaries pass through an optical cell filled with a buffer solution, and DNA fragments that migrate through the glass capillaries are analyzed using a laser beam. To minimize background light due to scattering, the glass capillaries are lined up in a single plane in a horizontal direction along the horizontally irradiated laser beam.
However, with such a conventional DNA analyzer, the glass capillaries have a circular cross section, and therefore the laser beam is scattered at the surfaces of the first glass capillary, resulting in it being impossible to irradiate all of the glass capillaries uniformly, that is, without the intensity of the laser beam dropping for subsequent glass capillaries.
To combat this problem, either the refractive index of the buffer solution in the optical cell is made to be the same as the refractive index of the glass capillaries (about 1.5), so that scattering of the laser beam at the surfaces of the glass capillaries is eliminated and all of the migration paths of the DNA fragments are irradiated with the laser beam, or else the portion of the capillary array corresponding to the region irradiated with the laser beam is removed so as to form buffer solution sheath flows. A device disclosed in Anal. Chem., 1994, Vol. 66, pages 1021-1026 provides an example of the latter.
FIG. 1 is a schematic view showing the structure of the DNA analyzer disclosed in the above-mentioned document.
In FIG. 1, twenty vertically oriented glass capillaries 11 are lined up in a single plane in a horizontal direction at a pitch of 0.35 mm in a sealed optical cell 10. Each of the glass capillaries 11 is comprised of a gel-filled capillary 12 that passes through a top wall of the optical cell 10, and an open capillary 13 that passes through a bottom wall of the optical cell 10. The bottom end of each gel-filled capillary 12 faces the top end of the corresponding open capillary 13, with a 1 mm gap in-between. A buffer solution 15 is fed into the optical cell 10 from a buffer vessel 14, and DNA fragments 16 are introduced into the gel-filled capillaries 12.
Due to the pressure of the buffer solution 15 in the buffer vessel 14, a sheath flow 17 of the buffer solution 15 is formed in the vicinity of the top end opening of each open capillary 13. When this sheath flow 17 flows into the open capillary 13, DNA fragments 16 are guided into the open capillary 13. A laser beam is irradiated horizontally onto the glass capillaries 11 at the level of the sheath flows 17, thus performing DNA analysis on the DNA fragments in the sheath flows 17. The laser beam used is, for example, an Ar+ 488 nm laser beam or a YAG 532 nm laser beam.
Quartz glass elements are used as the gel-filled capillaries 12 in such a DNA analyzer. As shown in FIG. 2, each quartz glass element has a square cross section (D=300 xcexcm), and has an internal hole having a square cross section (d=5 to 10 xcexcm).
However, with the method in which the refractive index of the buffer solution 15 in the optical cell 10 is made to be the same as the refractive index of the glass capillaries 11 (about 1.5), and the method in which a portion of each glass capillary 11 corresponding to the region irradiated with the laser beam is removed so as to cause a sheath flow 17 of the buffer solution 15 to be formed in each place where a portion of a glass capillary 11 has been removed, the DNA detection efficiency of the electrophoretic DNA analysis can be increased, but there are drawbacks, such as a special buffer solution having to be prepared, or the structure of the analyzer becoming complicated, with the gel-filled capillaries and the open capillaries having to be aligned with one another.
It is an object of the present invention to provide a glass capillary for DNA analysis which allows the DNA detection efficiency of a DNA analyzer that uses electrophoresis (hereinafter referred to as an xe2x80x98electrophoretic DNA analyzerxe2x80x99) to be increased, and allows the DNA analyzer to have a simple structure, a method of manufacturing the glass capillary, and a DNA analyzer that uses the glass capillary.
The inventors of the present invention have discovered that, if glass capillaries for DNA analysis that each have an internal hole wherein both the glass capillary and the internal hole have a rectangular cross section are used in an electrophoretic DNA analyzer, then scattering of the laser beam at the surfaces of the glass capillaries can be prevented and therefore the transmittance of the laser beam can be increased. As a result, the DNA detection efficiency of the electrophoretic DNA analyzer can be increased, and moreover the analyzer can be given a simple structure. Note that throughout this specification (including the claims), the term xe2x80x98rectangularxe2x80x99 includes xe2x80x98squarexe2x80x99; when xe2x80x98squarexe2x80x99 is not to be included, the term xe2x80x98oblongxe2x80x99 is used.
Moreover, the inventors of the present invention have discovered that, by making the internal hole of each glass capillary have an oblong cross section and placing each glass capillary such that the longitudinal axis of the internal hole therein extends along the direction of laser beam irradiation, thin-walled portions are formed in each glass capillary along the direction of the short sides of the cross section of the internal hole and hence absorption loss of the laser beam can be reduced, and moreover thick-walled portions are formed in each glass capillary along the direction of the long sides of the cross section of the internal hole and hence the strength of the glass capillary can be increased; the inventors of the present invention have also discovered that, by offsetting the internal hole in the direction of the short sides of the cross section thereof, a thin-walled portion is formed in the glass capillary along the direction of a long side of the cross section of the internal hole and hence absorption loss in the glass of the fluorescence from the DNA fragments caused by the laser beam irradiation can be reduced.
Furthermore, the inventors of the present invention have discovered that, if a mother glass having a through hole wherein each of the mother glass and the through hole has a rectangular cross section is prepared, and this mother glass is drawn while heating the same, then a glass capillary for DNA analysis can be easily produced, wherein the cross section of the glass capillary is approximately similar to but smaller than the cross section of the mother glass, and the cross section of the internal hole of the glass capillary is approximately similar to but smaller than the cross section of the through hole of the mother glass; the inventors of the present invention have also discovered that, if the heating temperature of the mother glass is such that the viscosity of the mother glass becomes 105 to 109 poise, then the glass capillary for DNA analysis and the internal hole thereof can both be given a rectangular cross section reliably and with good reproducibility.
To attain the above object, the present invention provides a glass capillary for DNA analysis having an internal hole, and each of the glass capillary and the internal hole has a rectangular cross section.
According to this glass capillary, since the glass capillary and the internal hole each have a rectangular cross section, scattering of the laser beam at the surface of the glass capillary can be prevented and hence the transmittance of the laser beam can be increased. As a result, the detection efficiency of the electrophoretic DNA analyzer can be increased, and moreover the analyzer can be given a simple structure.
Preferably, the internal hole has a cross-sectional area ratio of 25 to 90% relative to the glass capillary.
According to this glass capillary, a drop in the transmittance of the laser beam can be prevented while maintaining the strength of the glass capillary.
Preferably, the internal hole has an oblong cross section.
According to this glass capillary, thin-walled portions are formed in the glass capillary along the direction of the short sides of the cross section of the internal hole and hence absorption loss of the laser beam in the glass can be reduced, and moreover thick-walled portions are formed in the glass capillary along the direction of the long sides of the cross section of the internal hole and hence the strength of the glass capillary can be increased.
Also preferably, the internal hole is offset from a center of the glass capillary in a direction of short sides of the cross section of the internal hole.
According to this glass capillary, a thin-walled portion is formed in the glass capillary along the direction of a long side of the cross section of the internal hole and hence absorption loss in the glass of the fluorescence from DNA fragments caused by laser beam irradiation can be reduced. That is, the detection sensitivity of the label signals from the DNA fragments migrating through the glass capillary when detection is carried out from the thin-walled portion side of the glass capillary can be increased.
Preferably, the glass capillary comprises silicate glass having a total iron (Fe2O3 plus FeO) content of not more than 1000 ppm.
According to this glass capillary, absorption loss of the laser beam in the glass can be reduced. In particular, the absorption loss can be reduced when a laser beam of wavelength not more than 500 nm is used.
Preferably, the silicate glass contains not less than 45 wt % of SiO2.
According to this glass capillary, the transmission of light in the ultraviolet region can be improved, and the chemical resistance of the glass capillary to acids and alkalis can be improved.
More preferably, the silicate glass comprises 45 to 80 wt % of SiO2, 1 to 20 wt % of Al2O3, 5 to 30 wt % of RO (MgO, CaO, SrO, BaO, ZnO), and 4 to 14 wt %, of R2O (Na2O, K2O, Li2O).
According to this glass capillary, the chemical resistance of the glass capillary can be improved, and the formability of the glass capillary can be improved.
Still more preferably, the RO consists essentially of 0 to 8 wt % of MgO, 0 to 10 wt % of CaO, 0 to 10 wt % of SrO, 0 to 30 wt %, of BaO, and 0 to 4 wt % of ZnO.
To attain the above object, the present invention also provides a method of manufacturing a glass capillary for DNA analysis, comprising the steps of preparing a mother glass having a through hole, wherein each of the mother glass and the through hole has a rectangular cross section, and drawing the mother glass while heating the same.
According to this manufacturing method, a mother glass having a through hole wherein each of the mother glass and the through hole has a rectangular cross section is drawn while heating the same, and therefore, a glass capillary for DNA analysis can be easily produced, wherein the cross section of the glass capillary is approximately similar to but smaller than the cross section of the mother glass, and the cross section of the internal hole of the glass capillary is approximately similar to but smaller than the cross section of the through hole of the mother glass.
Preferably, the mother glass is heated to a temperature set to such a value that the mother glass has a viscosity of 105 to 109 poise.
According to this manufacturing method, the glass capillary for DNA analysis and the internal hole thereof can both be given a rectangular cross section reliably and with good reproducibility.
In a preferred form of the method according to the present invention, the step of preparing the mother glass comprises the steps of placing together four glass elements each having a polygonal cross section, and joining the glass elements together through heat fusion, thus producing the mother glass.
In another preferred form of the method, the step of preparing the mother glass comprises the steps of placing together four glass elements each having a polygonal cross section, and joining the glass elements together using an adhesive, thus producing the mother glass.
To attain the above object, the present invention further provides a DNA analyzer comprising an optical cell having a top wall and a bottom wall, a plurality of glass capillaries for DNA analysis mounted in the optical cell in a fashion passing through the top wall and the bottom wall, wherein each of the glass capillaries has a rectangular cross section, and each of the glass capillaries has an internal hole having a rectangular cross section, and wherein the glass capillaries are lined up in a single plane in a horizontal direction, and a device that feeds a fluid for DNA analysis into the optical cell.
According to this DNA analyzer, since each glass capillary and the internal hole thereof both have a rectangular cross section, scattering of the laser beam at the surfaces of the glass capillaries can be prevented and hence the transmittance of the laser beam can be increased, and as a result, the detection efficiency of the electrophoretic DNA analyzer can be increased, and moreover the analyzer can be given a simple structure.
Prefereably, the DNA analyzer according to the present invention comprises a device that irradiates the glass capillaries with a laser beam, and the internal hole of each of the glass capillaries has an oblong cross section, and wherein each of the glass capillaries is placed such that the internal hole thereof has a longitudinal axis thereof extending along a direction of irradiation of the laser beam.
According to this DNA analyzer, thin-walled portions are formed in each glass capillary along the direction of the short sides of the cross section of the internal hole thereof and hence absorption loss of the laser beam in the glass can be reduced, and moreover thick-walled portions are formed in each glass capillary along the direction of the long sides of the cross section of the internal hole thereof and hence the strength of the glass capillary can be increased.
The above and other objects, features and advantages of the invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings.